Urinary Trypsin Inhibitor (UTI), also known as ulinastatin, uristatin, urinastatin, ulistin, human inhibitor 30 (HI-30), mingin and bikunin, is a protease inhibitor with a molecular weight of about 40 kD. UTI is present in human urine and blood (hUTI) and has a variety of physiological activities such as an inhibitory effect on a family of serine proteases, such as trypsin, α-chymotrypsin, plasmin, cathepsin-G and leukocyte elastase. UTI also has immunomodulatory effect, and it can down-regulate the release of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1) and interleukin (IL-6). In addition, UTI also interferes with PDGF-D (PDGF-DD)/PDGF-BBR active dimer-mediated signaling pathway by neutralizing the dimer.
hUTI has received marketing authorization and one product is marketed in Japan under the trade name Miraclid and is isolated from human urine. In fact, hUTI isolated from human urine is currently marketed by several manufacturers for treatment of pancreatitis and acute circulatory failure caused by shock.
UTI is first produced in humans as a presursor protein called AMBP (α1-microglobulin/bikunin precursor), which is encoded on human chromosome 9. The proteolysis of AMBP yields the free UTI containing 143 amino acids. UTI comprises two Kunitz domains that are known to inhibit serine proteases, which are flanked by unstructured amino acids on UTI's N- and C-termini. The two domains are expected to confer differenct specificities of protease inhibition, due to the different amino acids involved in protease binding. By analogy to other serine protease inhibitors (e.g. BPTI, bovine pancreatic trypsin inhibitor), we can estimate that two key amino acids for protease inhibition include Met26 (Kunitz domain 1) and Arg88 (Kunitz domain 2). Little is know about the involvement of different portions of UTI during inhibition of different proteases, but removal of Kunitz domain 1 has been shown to change proteases specificity, uncovering new inhibitory activity against Factor Xa and plasma kallikrien. The full-length UTI does not show inhibition of these two proteases (Morishita et al., Thrombosis Research 1994, vol 73 (3/4) p 193-204). UTI also comprises two attached sugars, one O-linked at Ser10 and one N-linked at Asn45. The half-life of UTI in rodents and humans is 4-30 minutes (Fries et al, International Journal of Biochemistry and Cell Biology, 2000, vol 32, p 125-137).
A UTI fusion protein should contain optimized sequence of amino acids, including the best start and stop points of any UTI domains, and may be fused to another protein to enhance properties such as expression, purification, half-life, and stability. The exact sequence of the fusion partner needs determination and may include variations in linkers, start/stop points, and/or mutations that may change the functional properties of the fusion partner.
Variants of ulinastatin obtained from urine are known WO199856916, U.S. Pat. Nos. 5,792,629, 5,407,915, 5,409,895, 7,019,123, and 6,583,108. The concept of fusion proteins of ulinastatin (and variations thereof) has been disclosed U.S. 20080181892, U.S. Pat. No. 5,541,288, and U.S. 20080255025. Certain UTI fusion proteins are described in CN 103044554A. The fusion proteins of CN 103044554A relate to specific variants in the Fc domain, presumably to avoid any Fc mediated pharmacological effects (ADCC, CDC). We have surprisingly found that a UTI-Fc with wild type IgG1 is well tolerated and provides significant increase in half-life. Also, compared to the UTI fusion proteins of CN 103044554A the present UTI fusion proteins, in particular SEQ ID NO:1, demonstrate greater thermal stability.
The present invention provides UTI fusion proteins, pharmaceutical compositions comprising the same, preparation methods, and uses thereof.